Expression of highly immunogenic tuberculosis proteins in plants using a versatile Potato virus X-based expression system

The functionally and structurally-coordinated proteins ESAT-6 and CFP-10 secreted by Mycobacterium tuberculosis are potent T-cell antigens and hold great promise as novel subunit vaccines against tuberculosis (TB). Using a modified Potato virus X, we expressed the ESAT-6 and CFP-10 proteins in Nicotiana benthamiana and in tomato plants. Both proteins were tagged with a HiS(6)-Gly polypeptide at their N-termini, while a green fluorescent protein (GFP) was fused in-frame at their C-termini. Our data suggest that free ESAT-6 and CFP-10 were less stable than the GFP fusion proteins in plant cells. GFP-tagging significantly increased the stability of both proteins, as shown by the fact that ESAT6:GFP and CFP10:GFP were readily detected by western blotting. The GFP tag also allowed direct visualisation and localisation of green fluorescence, an efficient reporter of fusion protein expression in those plant tissues infected by the recombinant viruses. The potential for producing ESAT-6 and CFP-10 as novel orally-deliverable TB vaccines in tomato fruit is discussed